One of the major challenges in cancer immunotherapy is tumor-induced immune evasion. The mechanisms underlying this immune evasion include defective tumor-antigen CD4[+]CD25[+] regulatory T (T{Reg}) cells myeloablative chemotherapy with stem cell transplant may offer the best chance of achieving a state of minimal residual disease, and thus minimizing tumor-induced immune evasion. However, T{Reg}-mediated tumor-specific T cell tolerance persists following transplant. We have identified that in vivo stimulation of Toll-like receptors (TLRs) of the innate immunity is required for the reversal of T{Reg}-mediated suppression on tumor-specific CD8[+] T cells post-transplant. This can be achieved by conventional dendritic cell (cDC) vaccines co-administered with a TLR9 ligand, CpG in vivo. We have also discovered that vaccinia virus (W)-based vaccines can even more potently activate tumor-specific CDS T cell response post-transplant due to their unique ability to activate multiple innate immune pathways in vivo: W activates cDCs through TLR2, leading to the production of pro-inflammatory cytokines, and a TLR-independent pathway, resulting in secretion of type I interferons (IFNs);In addition, W also activates plasmacytoid DCs (pDCs) to secrete high levels of type I IFNs in a TLR-dependent manner. Taken together, these observations suggest a central hypothesis that efficient activation of innate immune system is critical for overcoming T{Reg}-mediated suppression on tumor-specific T cells through post-transplant vaccinations. To test this hypothesis, we propose to pursue the following four aims: 1) To determine if pDCs are critical for TLR9-dependent augmentation of anti-tumor CDS immunity post-transplant;2) to delineate the role of type I IFNs in TLR-dependent reversal of T{Reg}-mediated suppression on tumor-specific CD8[+] T cells post-transplant;3) To test if activation of multiple TLRs synergistically enhances the efficacy of post-transplant cDC vaccinations and generates long-term, protective anti-tumor immunity;4) To investigate the role of TLRs in promoting the differentiation of hematopoietic progenitor cells into pDCs and cDCs. We expect that this work will lead to the identification of critical factors for enhancing anti-tumor T cell immunity post-transplant, and in turn will help us design new generations of effective tumor vaccines in the settings of stem cell transplant.